Method for producing Fc-containing protein

Abstract

This specification provides an improved method for producing Fc-containing proteins. The method generally comprises culturing mammalian cells that produce Fc-containing proteins at a first pH setting for a first period of time, and then culturing the cells at a second pH setting higher than the first pH setting for a second period of time.

Description

Background Art

[0001] To produce a recombinant Fc-containing protein for therapeutic use, it generally involves the expression of the protein in cultured mammalian cells. The cell culture conditions can affect the yield and / or quality of the Fc-containing protein produced by the cultured cells. In particular, suboptimal cell culture conditions can lead to an increase in the amount of proteolytic partial cleavage (clipping) of Fc-containing proteins such as the produced dulaglutide, which can affect the yield and / or product quality.

[0002] Therefore, there is a need for an improved cell culture method for producing recombinant Fc-containing proteins that maximizes the protein yield and quality.

Summary of the Invention

[0003] The present disclosure provides an improved method for producing Fc-containing proteins. The method generally includes culturing mammalian cells expressing the Fc-containing protein at a first pH set value for a first period, and then culturing the cells at a second pH set value higher than the first pH set value for a second period. The method disclosed herein is particularly advantageous in that it can reduce the proteolytic partial cleavage of Fc-containing proteins such as dulaglutide.

[0004] In one aspect, a method for producing dulaglutide herein includes: a) culturing mammalian cells expressing dulaglutide in a cell culture medium at a first pH set value for a first period, and then b) culturing the mammalian cells in the cell culture medium at a second pH set value for a second period, wherein the second pH set value is higher than the first pH set value, and as a result, dulaglutide is produced by the mammalian cells.

[0005] In one embodiment, the first pH setting has a dead band of 0.01 to 0.10. In another embodiment, the first pH setting has a dead band of 0.07 to 0.10. In yet another embodiment, the first pH setting has a dead band of approximately 0.09.

[0006] In one embodiment, the second pH setting has a dead zone of 0.01 to 0.10. In another embodiment, the second pH setting has a dead zone of 0.03 to 0.06. In yet another embodiment, the second pH setting has a dead zone of approximately 0.05.

[0007] In one embodiment, the dead zone of the second pH setting is narrower than that of the first pH setting. In one embodiment, the first pH setting has a dead zone of approximately 0.09, and the second pH setting has a dead zone of approximately 0.05.

[0008] In one embodiment, the second pH setting is 0.01 to 1.0 pH units higher than the first pH setting. In one embodiment, the first pH setting is 6.0 to 7.0. In one embodiment, the first pH setting is 6.5 to 6.9. In one embodiment, the first pH setting is 6.8 to 6.9. In one embodiment, the first pH setting is approximately 6.86.

[0009] In one embodiment, the second pH setting is 6.5 to 7.5. In another embodiment, the second pH setting is 6.9 to 7.5. In yet another embodiment, the second pH setting is 7.0 to 7.1. In yet another embodiment, the second pH setting is approximately 7.0.

[0010] In one embodiment, the first pH setting value is approximately 6.86, with a dead zone of approximately 0.09, and the second pH setting value is approximately 7.0, with a dead zone of approximately 0.05.

[0011] In one embodiment, the first period is longer than the second period. In one embodiment, the first period is 7 to 12 days. In one embodiment, the first period is 9 to 11 days. In one embodiment, the first period is approximately 10 days.

[0012] In one embodiment, the second period is at least 1 day. In one embodiment, the second period is 4 to 7 days. In one embodiment, the second period is 4 to 6 days. In one embodiment, the second period is approximately 5 days.

[0013] In one embodiment, the first period is approximately 10 days, and the second period is approximately 5 days.

[0014] In one embodiment, the first pH setting value is approximately 6.86, its dead zone is approximately 0.09, and the first period is 9 to 10 days; the second pH setting value is approximately 7.0, its dead zone is approximately 0.05, and the second period is 4 to 5 days.

[0015] In one embodiment, the method further comprises culturing mammalian cells at a first temperature for 1 to 5 days, and then culturing the mammalian cells at a second temperature for 9 to 14 days. In one embodiment, the second temperature is lower than the first temperature.

[0016] In one embodiment, the first temperature is 34°C to 40°C. In one embodiment, the first temperature is approximately 36°C. In one embodiment, the second temperature is 30°C to 36°C. In one embodiment, the second temperature is approximately 33°C.

[0017] In one embodiment, mammalian cells are cultured at a first temperature for 3 to 4 days. In another embodiment, mammalian cells are cultured at a first temperature for approximately 3 days. In yet another embodiment, mammalian cells are cultured at a second temperature for 11 to 12 days. In yet another embodiment, mammalian cells are cultured at a second temperature for approximately 11 days.

[0018] In one embodiment, mammalian cells are cultured in a bioreactor. In another embodiment, mammalian cells are cultured in a fed-batch mode.

[0019] In one embodiment, the method further includes adding a base source to the cell medium if the pH falls below the dead zone during a first period, or adding an acid source to the cell medium if the pH rises above the dead zone. In one embodiment, the method further includes adding a base source to the cell medium if the pH falls below the dead zone during a second period, or adding an acid source to the cell medium if the pH rises above the dead zone. In one embodiment, the acid source is CO2. In one embodiment, the base source is a solution containing NaOH.

[0020] In one embodiment, the mammalian cell medium is spaded with air during a first period. In one embodiment, the mammalian cell medium is spaded with air during a second period. In one embodiment, the mammalian cell medium is spaded with air during both the first and second periods.

[0021] In one embodiment, the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cell is a CHO cell.

[0022] In one embodiment, one or more proteases have lower activity in a second pH dead zone compared to a first pH dead zone. In one embodiment, one or more proteases are present in cells or cell culture medium. In one embodiment, one or more proteases have lower activity at a second pH setting compared to a first pH setting. In one embodiment, one or more proteases partially cleave dulaglutide at the N-terminus. In one embodiment, the protease is cathepsin D. In one embodiment, less than approximately 4% of the dulaglutide produced is partially cleaved at the N-terminus.

[0023] In one aspect, provided herein is a dulaglutide produced by the methods disclosed herein.

[0024] Further embodiments of the invention are described below. 1. A method for producing an Fc-containing protein, comprising: a) culturing mammalian cells expressing the Fc-containing protein in a cell culture medium at a first pH setpoint for a first period, and then b) culturing the mammalian cells in the cell culture medium at a second pH setpoint for a second period, wherein the second pH setpoint is higher than the first pH setpoint, whereby the Fc-containing protein is produced by the mammalian cells. 2. The method according to embodiment 1, wherein the first pH setpoint has a dead band of 0.01 - 0.10. 3. The method according to embodiment 1 or 2, wherein the first pH setpoint has a dead band of 0.07 - 0.10. 4. The method according to any one of the preceding embodiments, wherein the first pH setpoint has a dead band of about 0.09. 5. The method according to any one of the preceding embodiments, wherein the second pH setpoint has a dead band of 0.01 - 0.10. 6. The method according to any one of the preceding embodiments, wherein the second pH setpoint has a dead band of 0.03 - 0.06. 7. The method according to any one of the preceding embodiments, wherein the second pH setpoint has a dead band of about 0.05. 8. The method according to any one of the preceding embodiments, wherein the dead band of the second pH setpoint is narrower than the dead band of the first pH setpoint. 9. The method according to any one of the preceding embodiments, wherein the first pH setpoint has a dead band of about 0.09 and the second pH setpoint has a dead band of about 0.05. 10. The method according to any one of the preceding embodiments, wherein the second pH setpoint is 0.01 - 1.0 pH units higher than the first pH setpoint. 11. The method according to any one of the preceding embodiments, wherein the first pH setpoint is 6.0 - 7.0. 12. The method according to any one of the preceding embodiments, wherein the first pH set value is 6.5 to 6.9. 13. The method according to any one of the preceding embodiments, wherein the first pH set value is 6.8 to 6.9. 14. The method according to any one of the preceding embodiments, wherein the first pH set value is about 6.86. 15. The method according to any one of the preceding embodiments, wherein the second pH set value is 6.5 to 7.5. 16. The method according to any one of the preceding embodiments, wherein the second pH set value is 6.9 to 7.5. 17. The method according to any one of the preceding embodiments, wherein the second pH set value is 7.0 to 7.1. 18. The method according to any one of the preceding embodiments, wherein the second pH set value is about 7.0. 19. The method according to any one of the preceding embodiments, wherein the first pH set value is about 6.86, the dead zone is about 0.09, the second pH set value is about 7.0, and the dead zone is about 0.05. 20. The method according to any one of the preceding embodiments, wherein the first period is longer than the second period. 21. The method according to any one of the preceding embodiments, wherein the first period is 7 to 12 days. 22. The method according to any one of the preceding embodiments, wherein the first period is 9 to 11 days. 23. The method according to any one of the preceding embodiments, wherein the first period is about 10 days. 24. The method according to any one of the preceding embodiments, wherein the second period is at least 1 day. 25. The method according to any one of the preceding embodiments, wherein the second period is 4 to 7 days. 26. The method according to any one of the preceding embodiments, wherein the second period is 4 to 6 days. 27. The method according to any one of the preceding embodiments, wherein the second period is about 5 days. 28. The method according to any one of the preceding embodiments, wherein the first period is about 10 days and the second period is about 5 days. 29. a) The first pH setting is approximately 6.86, the dead zone is approximately 0.09, the first period is 9-10 days, and b) The method according to any one of the prior embodiments, wherein the second pH setting is approximately 7.0, the dead zone is approximately 0.05, and the second period is 4 to 5 days. 30. The method according to any one of Embodiments 1 to 22, further comprising culturing mammalian cells at a first temperature for the first 1 to 5 days of a first period, and then culturing the mammalian cells at a second temperature for 9 to 14 days. 31. The method according to Embodiment 30, wherein the second temperature is lower than the first temperature. 32. The method according to Embodiment 30 or 31, wherein the first temperature is 34°C to 40°C. 33. The method according to any one of embodiments 30 to 32, wherein the first temperature is approximately 36°C. 34. The method according to any one of embodiments 30 to 33, wherein the second temperature is 30°C to 36°C. 35. The method according to any one of embodiments 30 to 34, wherein the second temperature is approximately 33°C. 36. The method according to any one of embodiments 30 to 35, wherein mammalian cells are cultured at a first temperature for 3 to 4 days. 37. The method according to any one of embodiments 30 to 36, wherein mammalian cells are cultured at a first temperature for approximately 3 days. 38. The method according to any one of embodiments 30 to 37, wherein mammalian cells are cultured at a second temperature for 11 to 12 days. 39. The method according to any one of embodiments 30 to 38, wherein mammalian cells are cultured at a second temperature for approximately 11 days. 40. The method according to any one of the prior embodiments, wherein cells are cultured in a bioreactor. 41. The method according to Embodiment 40, wherein mammalian cells are cultured in a fed-batch mode. 42. The method according to any one of the prior embodiments, wherein the mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. 43. The method according to any one of the prior embodiments, wherein the mammalian cells are CHO cells. 44. The method according to any one of the prior embodiments, further comprising adding a base source to the cell medium when the pH falls below the dead zone during the first period, or adding an acid source to the cell medium when the pH rises above the dead zone. 45. The method according to any one of the prior embodiments, further comprising adding a base source to the cell medium when the pH falls below the dead zone during the second period, or adding an acid source to the cell medium when the pH rises above the dead zone. 46. ​​The method according to embodiment 44 or 45, wherein the acid source is CO2. 47. The method according to Embodiment 44 or 45, wherein the base source is a solution containing NaOH. 48. The method according to any one of the prior embodiments, wherein the cell culture medium is spagged with air during a first period and / or a second period. 49. The method according to any one of the prior embodiments, wherein one or more proteases have lower activity in a second pH dead zone compared to a first pH dead zone. 50. The method according to any one of the prior embodiments, wherein one or more proteases have lower activity at a second pH setting compared to a first pH setting. 51. The method according to Embodiment 49 or 50, wherein one or more proteases partially cleave the Fc-containing protein at its N-terminus, and optionally the protease is cathepsin D. 52. The method according to any one of the prior embodiments, wherein the mammalian cell culture contains less than 4% of partially cleaved Fc-containing proteins at the end of the second period. 53. The method according to any one of the prior embodiments, wherein the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog having one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1). 54. The method according to any one of the prior embodiments, wherein the Fc-containing protein comprises a GLP-1 analog having the amino acid sequence of SEQ ID NO: 2. 55. The method according to any one of the prior embodiments, wherein the Fc-containing protein comprises a peptide linker. 56. The method according to Embodiment 55, wherein the peptide linker contains 1 to 10 G4S units (SEQ ID NO: 3). 57. The method according to Embodiment 55 or 56, wherein the peptide linker comprises the amino acid sequence described in SEQ ID NO: 3. 58. Fc-containing proteins a) A GLP-1 analog containing the amino acid sequence of SEQ ID NO: 2, b) A peptide linker containing the amino acid sequence of Sequence ID No. 3, c) A method according to any one of the prior embodiments, comprising the Fc portion of an immunoglobulin. 59. The method according to Embodiment 58, wherein the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc portion. The method according to any one of the prior embodiments, wherein the 60.Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 4. 61. The method according to any one of the prior embodiments, wherein the Fc-containing protein is a homodimer containing two identical amino acid chains, each containing the amino acid sequence of SEQ ID NO: 4. 62. The method according to any one of the prior embodiments, wherein the Fc-containing protein is dulaglutide. 63. A method for producing dulaglutide in CHO cells, a) A step of culturing CHO cells for about 10 days in a cell medium maintained at a first pH setting of approximately 6.68 with a dead zone of approximately 0.09, and thereafter, b) A step of culturing CHO cells for about 5 days in a cell medium maintained at a second pH setting of about 7.0 with a dead zone of about 0.05, The culture process is started at a temperature of approximately 36°C, and then reduced to approximately 33°C after about 3 days. As a result, dulaglutide is produced by CHO cells. 64. The method according to Embodiment 63, wherein CHO cells are cultured in a fed-batch mode in a bioreactor. 65. The method according to either Embodiment 63 or 64, wherein NaOH is added to the cell culture medium when the pH falls below the dead zone of either the first pH setting or the second pH setting, and CO2 is added to the cell culture medium when the pH rises above the dead zone of either the first pH setting or the second pH setting. 66. The method according to any one of embodiments 63 to 65, wherein the cell culture medium is spared with air once or more times. 67. The method according to any one of embodiments 63 to 67, wherein less than 4% of the dulaglutide produced by CHO cells is partially cleaved at the N-terminus. 64. An Fc-containing protein produced by the method described in any one of the prior embodiments. [Brief explanation of the drawing]

[0025] [Figure 1] This graph shows the pCO2 levels in the production bioreactor over 14 days of culture in two different experiments at pH 6.80±0.03 or two different experiments at pH 6.86±0.09. [Figure 2] This plot shows the pH and desH / HG levels on day 14 in 3L and 5L production bioreactors. [Modes for carrying out the invention]

[0026] This disclosure provides an improved method for producing Fc-containing proteins. The method generally includes culturing mammalian cells expressing Fc-containing proteins at a first pH setting for a first period, and then culturing the cells at a second pH setting higher than the first pH setting for a second period. The method disclosed herein is particularly advantageous in that it can reduce partial cleavage of Fc-containing proteins by proteases.

[0027] I. Definition As used herein, the term “pH setpoint” refers to the desired pH value of the cell medium. In certain embodiments, the pH setpoint of the cell medium is actively maintained in response to pH changes that fall outside a designated dead zone (e.g., by adding an acid or base to the cell medium).

[0028] As used herein, the term “dead band” refers to the range of pH values ​​above and below a pH setpoint in which the pH of a cell medium is allowed to fluctuate without intervention. For example, a cell medium with a pH setpoint of 7.00 and a dead band of 0.05 may have a pH of 6.95 to 7.05. Generally, the pH setpoint and dead band are expressed as “setpoint ± dead band,” for example, 7.00 ± 0.05.

[0029] As used herein, the terms "pH shift" or "pH setpoint shift" refer to a change in the pH of a cell culture medium caused by actively changing the pH setpoint to a lower or higher value.

[0030] As used herein, the term “Fc-containing protein” refers to a protein containing an Fc region. In one embodiment, the Fc-containing protein contains a variant Fc region comprising one or more amino acid substitutions, additions, and / or deletions to a naturally occurring Fc region. In one embodiment, the Fc-containing protein is an antibody. In one embodiment, the Fc-containing protein is not an antibody.

[0031] As used herein, the term “antibody” includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies, though not limited to these, include monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain and antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs(scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and any of the above antigen-binding fragments.

[0032] As used herein, the term “approximately” includes a variation of ±5% of the value or parameter specified herein. For example, when referring to a pH value, “approximately” means a range that includes values ​​5% lower and 5% higher than the reference value. Therefore, a pH of approximately 10 means a pH that encompasses pH 9.5 to pH 10.5.

[0033] II. Methods for culturing mammalian cells When producing Fc-containing proteins, optimizing the pH conditions of the mammalian cell culture in the production bioreactor is important for maintaining the quality of the Fc-containing protein product. This specification discloses a method for producing Fc-containing proteins, comprising culturing mammalian cells expressing Fc-containing proteins at a first pH setting, and then shifting to a second pH setting higher than the first pH setting. The method disclosed herein is particularly advantageous in that it can reduce the partial cleavage of Fc-containing proteins by proteases in the mammalian cell culture, thereby increasing the amount of intact Fc-containing proteins.

[0034] In one embodiment, the method disclosed herein generally comprises a) culturing mammalian cells expressing an Fc-containing protein in a cell medium at a first pH setting for a first period of time, and then b) culturing the mammalian cells in a cell medium at a second pH setting for a second period of time, wherein the second pH setting is higher than the first pH setting, and as a result, the Fc-containing protein is produced by the mammalian cells.

[0035] In one embodiment, the method disclosed herein generally comprises a) culturing mammalian cells expressing dulaglutide in a cell medium at a first pH setting for a first period of time, and then b) culturing the mammalian cells in a cell medium at a second pH setting for a second period of time, wherein the second pH setting is higher than the first pH setting, and as a result, dulaglutide is produced by the mammalian cells.

[0036] An exemplary cell culture method and conditions suitable for use in the above-described method are described in detail below.

[0037] In one embodiment, the first pH setting has a dead zone of 0.01 to 0.10. In one embodiment, the first pH setting has a dead zone of 0.07 to 0.12. In one embodiment, the first pH setting has a dead zone of 0.07 to 0.10. In one embodiment, the first pH setting is approximately 0.01, approximately 0.02, approximately 0.03, approximately 0.04, approximately 0.05, approximately 0.06, approximately 0.07, approximately 0.08, approximately 0.09, approximately 0.1, approximately 0.11, approximately 0.12, approximately 0.13, approximately 0.14, or approximately 0.15. In one embodiment, the first pH setting has a dead zone of approximately 0.09.

[0038] In one embodiment, the second pH setting has a dead zone of 0.01 to 0.10. In one embodiment, the second pH setting has a dead zone of 0.03 to 0.08. In one embodiment, the second pH setting has a dead zone of 0.03 to 0.06. In one embodiment, the second pH setting is approximately 0.01, approximately 0.02, approximately 0.03, approximately 0.04, approximately 0.05, approximately 0.06, approximately 0.07, approximately 0.08, approximately 0.09, approximately 0.1, approximately 0.11, approximately 0.12, approximately 0.13, approximately 0.14, or approximately 0.15. In one embodiment, the second pH setting has a dead zone of approximately 0.05.

[0039] In one embodiment, the dead zone of the second pH setting is narrower than that of the first pH setting. In one embodiment, the first pH setting has a dead zone of approximately 0.09, and the second pH setting has a dead zone of approximately 0.05.

[0040] In one embodiment, the second pH setting is 0.01 to 1.0 pH units higher than the first pH setting. In one embodiment, the second pH setting is approximately 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 pH unit higher than the first pH setting. In one embodiment, the second pH setting is 0.1 to 0.2 pH units higher than the first pH setting. In one embodiment, the second pH setting is approximately 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 pH units higher than the first pH setting.

[0041] In one embodiment, the first pH setting is 6.0 to 7.0. In one embodiment, the first pH setting is 6.5 to 6.9. In one embodiment, the first pH setting is 6.8 to 6.9. In one embodiment, the first pH setting is 6.85 to 7.0. In one embodiment, the first pH setting is approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, or approximately 6.9. In one embodiment, the first pH setting is approximately 6.86.

[0042] In one embodiment, the first pH setting is approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, and the dead zone is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15. In one embodiment, the first pH setting is approximately 6.86, and the dead zone is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15.

[0043] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.01.

[0044] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.02.

[0045] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.03.

[0046] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.04.

[0047] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.05.

[0048] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.06.

[0049] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.07.

[0050] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.08.

[0051] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.09.

[0052] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.1.

[0053] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.11.

[0054] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.12.

[0055] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.13.

[0056] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.14.

[0057] In one embodiment, the first pH setting values ​​are approximately 6.8, 6.81, 6.82, 6.83, 6.84, 6.85, 6.86, 6.87, 6.88, 6.89, or 6.9, with a dead zone of approximately 0.15.

[0058] In one embodiment, the second pH setting is 6.5 to 7.5. In one embodiment, the second pH setting is 6.9 to 7.5. In one embodiment, the second pH setting is 6.9 to 7.1. In one embodiment, the second pH setting is 7.0 to 7.1. In one embodiment, the second pH setting is approximately 6.9, approximately 6.9, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7.0, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7.04, approximately 7.05, approximately 7.06, approximately 7.07, approximately 7.08, approximately 7.09, or approximately 7.1. In one embodiment, the second pH setting is approximately 7.0.

[0059] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15. In one embodiment, the second pH setting is approximately 7.0, and its dead zone is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15.

[0060] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.01.

[0061] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.02.

[0062] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.03.

[0063] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.04.

[0064] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.05.

[0065] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.06.

[0066] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.07.

[0067] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.08.

[0068] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.09.

[0069] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.1.

[0070] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.11.

[0071] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.12.

[0072] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.13.

[0073] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.14.

[0074] In one embodiment, the second pH setting values ​​are approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1, with a dead zone of approximately 0.15.

[0075] In one embodiment, the first pH setting is 6.0 to 7.0, and the second pH setting is approximately 7.0. In one embodiment, the first pH setting is 6.5 to 6.9, and the second pH setting is approximately 7.0. In one embodiment, the first pH setting is 6.8 to 6.9, and the second pH setting is approximately 7.0. In one embodiment, the first pH setting is 6.85 to 7.0, and the second pH setting is approximately 7.0. In one embodiment, the first pH setting is approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, and approximately 6.9, and the second pH setting is approximately 7.0.

[0076] In one embodiment, the first pH setting is approximately 6.86 and the second pH setting is 6.5 to 7.5. In another embodiment, the first pH setting is approximately 6.86 and the second pH setting is 6.9 to 7.5. In yet another embodiment, the first pH setting is approximately 6.86 and the second pH setting is 6.9 to 7.1. In yet another embodiment, the first pH setting is approximately 6.86 and the second pH setting is 7.0 to 7.1. In one embodiment, the first pH setting is approximately 6.86, and the second pH setting is approximately 6.9, 6.9, 6.92, 6.93, 6.94, 6.95, 6.96, 6.97, 6.98, 6.99, 7.0, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7.07, 7.08, 7.09, or 7.1. In one embodiment, the first pH setting is approximately 6.86, and the second pH setting is approximately 7.0.

[0077] In one embodiment, the first pH setting value is approximately 6.86, with a dead zone of approximately 0.09, and the second pH setting value is approximately 7.0, with a dead zone of approximately 0.05.

[0078] In one embodiment, the first period is longer than the second period. In one embodiment, the first period is 7 to 12 days. In one embodiment, the first period is 9 to 11 days. In one embodiment, the first period is about 10 days. In one embodiment, the first period is about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, or about 12 days. In one embodiment, the first and second periods together are about 14 days.

[0079] In one embodiment, the second period is at least one day. In one embodiment, the second period is four to seven days. In one embodiment, the second period is four to six days. In one embodiment, the second period is approximately four days, five days, six days, or seven days. In one embodiment, the second period is approximately five days.

[0080] In one embodiment, the first period is approximately 8 days, and the second period is at least 1 day. In one embodiment, the first period is approximately 8 days, and the second period is 4 to 7 days. In one embodiment, the first period is approximately 8 days, and the second period is 4 to 6 days. In one embodiment, the first period is approximately 8 days, and the second period is approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0081] In one embodiment, the first period is approximately 9 days and the second period is at least 1 day. In one embodiment, the first period is approximately 9 days and the second period is 4 to 7 days. In one embodiment, the first period is approximately 9 days and the second period is 4 to 6 days. In one embodiment, the first period is approximately 9 days and the second period is approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0082] In one embodiment, the first period is approximately 10 days, and the second period is at least 1 day. In one embodiment, the first period is approximately 10 days, and the second period is 4 to 7 days. In one embodiment, the first period is approximately 10 days, and the second period is 4 to 6 days. In one embodiment, the first period is approximately 10 days, and the second period is approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0083] In one embodiment, the first period is approximately 11 days, and the second period is at least 1 day. In one embodiment, the first period is approximately 11 days, and the second period is 4 to 7 days. In one embodiment, the first period is approximately 11 days, and the second period is 4 to 6 days. In one embodiment, the first period is approximately 11 days, and the second period is approximately 4 days, approximately 5 days, approximately 6 days, or approximately 7 days.

[0084] In one embodiment, the first period is approximately 11 days and the second period is approximately 4 days. In one embodiment, the first period is approximately 11 days and the second period is approximately 3 days. In one embodiment, the first period is approximately 10 days and the second period is approximately 5 days. In one embodiment, the first period is approximately 10 days and the second period is approximately 4 days. In one embodiment, the first period is approximately 9 days and the second period is approximately 6 days. In one embodiment, the first period is approximately 9 days and the second period is approximately 5 days.

[0085] In one embodiment, the first pH setting is 6.8 to 6.9, its dead zone is 0.08 to 0.1, and the first period is 9 to 10 days; the second pH setting is 7.0 to 7.1, its dead zone is 0.04 to 0.06, and the second period is 4 to 5 days.

[0086] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 9 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 6 days.

[0087] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 9 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 5 days.

[0088] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 10 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 5 days.

[0089] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 10 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 4 days.

[0090] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 11 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 4 days.

[0091] In one embodiment, the first pH setting is 6.8 to 6.9, the dead zone is 0.08 to 0.1, and the first period is approximately 11 days; the second pH setting is 7.0 to 7.1, the dead zone is 0.04 to 0.06, and the second period is approximately 3 days.

[0092] In one embodiment, the first pH setting is approximately 6.86, its dead zone is approximately 0.09, and the first period is 9 to 11 days; the second pH setting is approximately 7.0, its dead zone is approximately 0.05, and the second period is 4 to 6 days.

[0093] In one embodiment, the first pH setting is approximately 6.86, its dead zone is approximately 0.09, and the first period is 9 to 11 days; the second pH setting is approximately 7.0, its dead zone is approximately 0.05, and the second period is approximately 4 days.

[0094] In one embodiment, the first pH setting is approximately 6.86, its dead zone is approximately 0.09, and the first period is 9 to 11 days; the second pH setting is approximately 7.0, its dead zone is approximately 0.05, and the second period is approximately 5 days.

[0095] In one embodiment, the first pH setting is approximately 6.86, its dead zone is approximately 0.09, and the first period is 9 to 11 days; the second pH setting is approximately 7.0, its dead zone is approximately 0.05, and the second period is approximately 6 days.

[0096] In one embodiment, the method further comprises culturing mammalian cells at a first temperature for 1 to 5 days, followed by culturing mammalian cells at a second temperature for 9 to 14 days. In one embodiment, the method further comprises culturing mammalian cells at a first temperature for about 1 day, followed by culturing mammalian cells at a second temperature for about 9, 10, 11, 12, 13, or 14 days. In one embodiment, the method further comprises culturing mammalian cells at a first temperature for about 2 days, followed by culturing mammalian cells at a second temperature for about 9, 10, 11, 12, 13, or 14 days. In one embodiment, the method further comprises culturing mammalian cells at a first temperature for about 3 days, followed by culturing mammalian cells at a second temperature for about 9, 10, 11, 12, 13, or 14 days. In one embodiment, the method further comprises culturing mammalian cells at a first temperature for about 4 days, and then culturing the mammalian cells at a second temperature for about 9, 10, 11, 12, 13, or 14 days. In one embodiment, the method further comprises culturing mammalian cells at a first temperature for about 5 days, and then culturing the mammalian cells at a second temperature for about 9, 10, 11, 12, 13, or 14 days.

[0097] In one embodiment, the second temperature is lower than the first temperature. In one embodiment, the second temperature is about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or about 6°C lower than the first temperature.

[0098] In one embodiment, the first temperature is 34°C to 40°C. In one embodiment, the first temperature is approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, or approximately 40°C. In one embodiment, the first temperature is approximately 36°C.

[0099] In one embodiment, the second temperature is 30°C to 36°C. In one embodiment, the second temperature is approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, or approximately 36°C. In one embodiment, the second temperature is approximately 33°C.

[0100] In one embodiment, mammalian cells are cultured at a first temperature for 3 to 4 days. In another embodiment, mammalian cells are cultured at a first temperature for approximately 3 days. In yet another embodiment, mammalian cells are cultured at a second temperature for 11 to 12 days. In yet another embodiment, mammalian cells are cultured at a second temperature for approximately 11 days.

[0101] In one embodiment, mammalian cells are cultured in a bioreactor. In another embodiment, mammalian cells are cultured in a fed-batch mode.

[0102] Examples of bioreactors, though not limited to these, include plug-flow bioreactors, continuous agitated tank bioreactors, fixed-bed bioreactors, air-lift bioreactors, and bioreactor bags.

[0103] Methods for adjusting the pH in a bioreactor are generally well known in the art. In one embodiment, the method further includes adding a base source to the cell medium if the pH falls below the dead zone during a first period, or adding an acid source to the cell medium if the pH rises above the dead zone. In one embodiment, the method further includes adding a base source to the cell medium if the pH falls below the dead zone during a second period, or adding an acid source to the cell medium if the pH rises above the dead zone. In one embodiment, the acid source is CO2. In one embodiment, the base source is a solution containing NaOH.

[0104] In one embodiment, the mammalian cell medium is sparged with air during a first period. In one embodiment, the mammalian cell medium is sparged with air during a second period. In one embodiment, the mammalian cell medium is sparged with air during both the first and second periods. In one embodiment, the mammalian cell medium is sparged with air during the first and / or second period to reduce the pCO2 in the cell medium. In one embodiment, the mammalian cell medium is sparged with air to increase the pH.

[0105] In one embodiment, the mammalian cell is selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cell is a CHO cell.

[0106] The cell culture media used in the methods disclosed herein may include any medium suitable for culturing mammalian cells, which are well known to those skilled in the art.

[0107] In one embodiment, one or more proteases have lower activity in a second pH dead zone compared to a first pH dead zone. In one embodiment, one or more proteases have lower activity at a second pH setting compared to a first pH setting. In one embodiment, one or more proteases are present in cells or cell culture medium. In one embodiment, one or more proteases partially cleave dulaglutide at the N-terminus, and optionally, the protease is cathepsin D.

[0108] In one embodiment, less than 4% of the dulaglutide produced is partially cleaved at the N-terminus. In one embodiment, less than 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, and 2.5% of the dulaglutide produced is partially cleaved at the N-terminus. In one embodiment, the dulaglutide partially cleaved at the N-terminus does not have either the N-terminal H1 or G2 residue of dulaglutide.

[0109] In one embodiment, less than 4% of the Fc-containing protein produced is partially cleaved at the N-terminus. In one embodiment, less than 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, or less than 1.0% of the Fc-containing protein produced is partially cleaved at the N-terminus.

[0110] III.Fc-containing protein The method provided herein is for producing Fc-containing proteins by culturing mammalian cells that express Fc-containing proteins.

[0111] In one embodiment, the Fc-containing protein includes one or more of the amino acid sequences listed in Table 1 below.

[0112] In one embodiment, the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog that includes one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0113] In one embodiment, the Fc-containing protein includes a GLP-1 analog having the amino acid sequence of SEQ ID NO: 2.

[0114] In one embodiment, the Fc-containing protein includes a peptide linker. In one embodiment, the C-terminal amino acid of the GLP-1 analog portion of the Fc-containing protein is fused to the N-terminus of the Fc portion of the immunoglobulin via the peptide linker. In one embodiment, the peptide linker contains 1 to 10 G4S units (SEQ ID NO: 3).

[0115] In one embodiment, the Fc-containing protein comprises a GLP-1 analog containing the amino acid sequence of SEQ ID NO: 2, a peptide linker (SEQ ID NO: 3) containing 1 to 10 G4S units, and the Fc portion of the immunoglobulin. In one embodiment, the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc portion.

[0116] In one embodiment, the Fc-containing protein contains the amino acid sequence of SEQ ID NO: 4. In one embodiment, the Fc-containing protein is a homodimer containing two identical amino acid chains, each containing the amino acid sequence of SEQ ID NO: 4.

[0117] In one embodiment, the Fc-containing protein is dulaglutide.

[0118] Dulaglutide is a human GLP-1 receptor agonist, comprising a dimer of a GLP-1 analog fused to the C-terminus of the Fc portion of an immunoglobulin analog via a (G4S)3 peptide linker, and is identified as CAS registry number 923950-08-7, with the following chemical name: 7-37-glucagon-like peptide 1 [8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) and peptide (synthetic 16-amino acid linker), fusion protein with immunoglobulin G4 (synthetic human Fc fragment), dimer. Each monomer of dulaglutide has the amino acid sequence described in SEQ ID NO: 4.

[0119] Two monomers are linked by a disulfide bond between cysteine ​​residues at positions 55 and 58 of SEQ ID NO: 4 to form a dimer. The structure, function, production, and use of dulaglutide in the treatment of T2DM are described in detail in U.S. Patent No. 7,452,966 and U.S. Patent Application Publication No. 2010 / 0196405. Dulaglutide has been shown to act as an agonist on the GLP-1 receptor, stimulating insulin synthesis and secretion, and providing improved glycemic control in patients with T2DM.

[0120] As used herein, the term “dulaglutide” means any GLP-1 receptor agonist protein dimer consisting of two monomers having the amino acid sequence of SEQ ID NO: 4, and includes any protein that is the subject of a regulatory submission seeking approval of a GLP-1 receptor agonist product, relying in whole or in part on data relating to dulaglutide submitted to regulatory authorities by Eli Lilly and Company, regardless of whether the party seeking approval of the protein actually identifies the protein as dulaglutide or uses any other term.

[0121] [Table 1]

[0122] In one embodiment, the Fc-containing protein is etanercept, alefacept, abatacept, lilonacept, romiprostim, belatacept, aflibercept, convercept, efmoloctocog alfa, eftrenonacog alfa, asfotase alfa, or ruspatacept.

[0123] In one embodiment, the Fc-containing protein is an antibody. In another embodiment, the Fc-containing protein is not an antibody.

[0124] In one embodiment, the Specified herein provides an Fc-containing protein produced by any one of the methods disclosed herein.

[0125] In one embodiment, a dulaglutide produced by any one of the methods disclosed herein is provided. [Examples]

[0126] The following examples are provided for illustrative purposes only and not for limitation.

[0127] Example 1. Analysis of high pCO2 levels and pH setpoint shift in a production bioreactor This example describes a study in which changes in the initial pH setpoint and dead zone were evaluated, and shifts in the pH setpoint and dead zone were performed during the production bioreactor stage of dulaglutide production, in order to identify parameters that reduce the pCO2 level in the product and minimize the level of partial cleavage morphs of dulaglutide (e.g., dulaglutide variants in which the H1 residue or both H1 and G2 residues are cleaved from the N-terminus of dulaglutide, such as the N-terminal cleavage species, desH / HG). In the dulaglutide production scheme, the initial pH setpoint and dead zone at the production bioreactor stage were set to 6.80 ± 0.03. However, under these conditions, an increase in pCO2 in the cell culture resulted in an increase in the partial cleavage morphs of dulaglutide, and therefore a decrease in dulaglutide titer.

[0128] Initial studies evaluating changes to the initial pH setting and the dead zone to 6.86±0.09 showed that changing the initial pH setting and widening the dead zone reduced the high pCO2 levels observed under conventional culture conditions during the production bioreactor stage (Figure 1). Therefore, under the tested conditions, pCO2 was maintained at a lower level with an initial pH of 6.86±0.09. The following small-scale studies investigate whether adding a pH shift can further optimize the cell culture conditions at this stage of dulaglutide production.

[0129] These small-scale studies were conducted using the current dulaglutide working cell bank (WCB), raw materials, process schedule, and process conditions that best replicate the commercial dulaglutide production cell culture process when implemented on a laboratory scale. The test conditions focused particularly on pH control during the production bioreactor phase of dulaglutide manufacturing.

[0130] The objective of the following experiment was to run CO2 clamp software to reproduce high pCO2 levels and to evaluate the effect of the pH shift on day 11 on the des H / HG levels. The conditions for this experiment are described in Table 2 below.

[0131] [Table 2]

[0132] The CO2 clamp software for the Applikon 3L bioreactor systems used in these tests operated via an internal computer script that calculated the CO2 gas flow based on a percentage of the total gas flow (air sparge + O2 sparge) and manual input of specific pCO2 target increase settings. The CO2 clamp automatically shuts off when the process moves outside the pH dead zone, thereby maintaining pH through pH control (either a CO2 sparging cascade or 2N NaOH). For example, if the baseline pCO2 without additional CO2 sparging was 30 mmHg and the CO2 clamp software was set to 5 mmHg, the pCO2 was expected to increase to 35 mmHg.

[0133] The viable cell density (VCD) over a 14-day culture period under four different conditions was comparable across all tested conditions. Cell viability remained similar under each condition until the end of the culture, although the CO2 clamp condition resulted in a slight decrease in cell viability. Glucose concentrations in the cell medium were comparable under each condition throughout the 14-day culture, requiring a pH shift to 7.00±0.05 on day 11 and glucose supply on day 13 to prevent glucose depletion before harvesting on day 14. Under the conditions with CO2 clamp and a pH shift to 6.75±0.02, a slight increase in lactate concentration in the cell medium was observed from day 11 to the end of the 14-day culture; without these measures, lactate concentration in the medium was comparable.

[0134] Sodium levels in the cultures were comparable under all conditions throughout the 14 days, and the offline pH profiles were highly comparable until the pH shift on day 11. Under CO2 clamp conditions, pCO2 (n=8) gradually increased after day 6 until approximately day 10, at which point the average pCO2 leveled off at approximately 70 mmHg until the end of the experiment. Dulaglutide titers were comparable under CO2 clamp conditions, except that the average titer on day 14 was lower.

[0135] Of the four conditions tested in this study, the CO2 clamp condition resulted in the lowest average titer and the greatest overall variability, which was attributed to the lactate concentration observed with this combination of bioreactor conditions (i.e., increased lactate led to a decrease in titer) (Table 3). The shift to a pH setting of 7.00 on day 11 resulted in the highest average titer.

[0136] [Table 3]

[0137] The CO2 clamp condition resulted in an average desH / HG value of 3.36%, with several data points exceeding the acceptable threshold of 3.5%. Furthermore, a shift in the pH setting to 6.75 on day 11 (condition 4) resulted in an average desH / HG value of 3.20%, with two data points close to or exceeding the acceptable threshold. However, a shift in the pH setting to 7.00 on day 11 (condition 3) resulted in the lowest average desH / HG value of 2.27% (Table 4).

[0138] [Table 4]

[0139] A positive correlation was observed between pCO2 and desH / HG on day 14. A negative correlation was observed between offline pH and desH / HG on day 14.

[0140] The results of this study showed that a higher pCO2 or a shift to a lower pH setting on day 11 correlated with higher levels of des H / HG and lower dulaglutide titer, and a shift to a higher pH setting on day 11 correlated with lower levels of des H / HG and higher dulaglutide titer.

[0141] Example 2. Identification of optimal pH conditions in the production bioreactor. This study was designed to identify the optimal pH strategy at the production bioreactor stage of the dulaglutide production scheme, including the days for pH shifts, pH setpoints and dead zone shift magnitudes, and pCO2 levels for process stability and critical product quality attributes (specifically, the level of the partial cleavage form of dulaglutide (des H / HG)). The conditions used in this study are listed in Table 5 below. All conditions were tested in a 5L production bioreactor for a total of 14 days.

[0142] In these tests, CO2 control in the Sartorius 5L system was manually controlled by calculating the CO2 gas flow setpoint daily using the total gas flow (air sparge + O2 sparge). Please refer to the formula below.

[0143]

number

[0144] [Table 5]

[0145] Viable cell density and viability levels were similar under all conditions throughout the 14-day culture period, with a slight decrease in viability during the last two days in the conditions where an increase in lactate was observed. Glucose concentrations in the cell medium were similar up to the pH shift on days 8, 9, and 10, and a general increase in glucose consumption was observed in cultures with a pH shift of 7.07 ± 0.05. An increase in lactate concentration in the medium towards the end of the 14 days was observed in all tanks with high pCO2.

[0146] The results of this study showed that offline pH profiles and sodium levels remained similar up to days 8, 9, and 10, when different pH shifts were performed. Dulaglutide titers were similar on day 8 but became lower by day 14. The lowest titers were observed in the bioreactor where high pCO2 and high lactate levels were also observed.

[0147] Analysis of desH / HG levels showed that the pH level of the production bioreactor on day 14 was negatively correlated with the desH / HG level in the production bioreactor test (Figure 2), suggesting that a higher pH setpoint and a pH shift to a narrower dead zone to maintain a higher pH towards the end of day 14 is the optimal pH control strategy for the production bioreactor phase of dulaglutide production in this test.

[0148] Further analysis of these test results showed a statistically significant negative correlation between the pH shift setpoint and desH / HG levels (scaled correlation estimate = -0.25, p = 0.0363), indicating that shifting to a higher pH setpoint reduced the amount of partial protease cleavage in the culture. Furthermore, a statistically significant negative correlation was found between pCO2 and dulaglutide recovery titer (scaled correlation estimate = -0.154, p = 0.0077), suggesting that increased pCO2 may result in lower dulaglutide titer in the bioreactor culture. The results also showed minimal influence on or correlation with dulaglutide titer or desH / HG levels on the day of the pH shift (days 8, 9, or 10).

[0149] In summary, the tests described in Examples 1 and 2 demonstrated that an initial pH setting and dead zone of 6.86±0.09 mitigated the increase in pCO2 observed at a pH setting and dead zone of 6.80±0.03. Furthermore, shifting to a higher pH setting and a narrower dead zone on days 8, 9, 10, or 11 of cell culture resulted in reduced desH / HG levels and increased dulaglutide titer compared to cell cultures where the initial pH setting and dead zone were maintained throughout the entire culture.

[0150] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention other than those described will be apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications shall be included within the scope of the accompanying claims.

[0151] Other embodiments are included in the following claims.

Claims

1. A method for producing dulaglutide, a) A step of culturing mammalian cells expressing the dulaglutide in a cell medium at a first pH setting for a first period of time, and thereafter, b) A step of culturing the mammalian cells in the cell medium at a second pH setting value for a second period of time, wherein the second pH setting value is higher than the first pH setting value. A method wherein the dulaglutide is produced by the mammalian cells as a result.

2. The method according to claim 1, wherein the first pH setting has a dead zone of 0.01 to 0.

10.

3. The method according to claim 1 or 2, wherein the first pH setting has a dead zone of 0.07 to 0.

10.

4. The method according to any one of claims 1 to 3, wherein the first pH setting has a dead zone of about 0.

09.

5. The method according to any one of claims 1 to 4, wherein the second pH setting has a dead zone of 0.01 to 0.

10.

6. The method according to any one of claims 1 to 5, wherein the second pH setting has a dead zone of 0.03 to 0.

06.

7. The method according to any one of claims 1 to 6, wherein the second pH setting has a dead zone of about 0.

05.

8. The method according to any one of claims 1 to 7, wherein the dead zone of the second pH setting value is narrower than the dead zone of the first pH setting value.

9. The method according to any one of claims 1 to 8, wherein the first pH setting has a dead zone of about 0.09 and the second pH setting has a dead zone of about 0.

05.

10. The method according to any one of claims 1 to 9, wherein the second pH setting value is 0.01 to 1.0 pH units higher than the first pH setting value.

11. The method according to any one of claims 1 to 10, wherein the first pH setting value is 6.0 to 7.

0.

12. The method according to any one of claims 1 to 11, wherein the first pH setting value is 6.5 to 6.

9.

13. The method according to any one of claims 1 to 12, wherein the first pH setting value is 6.8 to 6.

9.

14. The method according to any one of claims 1 to 13, wherein the first pH setting value is approximately 6.

86.

15. The method according to any one of claims 1 to 14, wherein the second pH setting value is 6.5 to 7.

5.

16. The method according to any one of claims 1 to 15, wherein the second pH setting value is 6.9 to 7.

5.

17. The method according to any one of claims 1 to 16, wherein the second pH setting value is 7.0 to 7.

1.

18. The method according to any one of claims 1 to 17, wherein the second pH setting value is approximately 7.

0.

19. The method according to any one of claims 1 to 18, wherein the first pH setting value is approximately 6.86 and the dead zone is approximately 0.09, and the second pH setting value is approximately 7.0 and the dead zone is approximately 0.

05.

20. The method according to any one of claims 1 to 19, wherein the first period is longer than the second period.

21. The method according to any one of claims 1 to 20, wherein the first period is 7 to 12 days.

22. The method according to any one of claims 1 to 21, wherein the first period is 9 to 11 days.

23. The method according to any one of claims 1 to 22, wherein the first period is approximately 10 days.

24. The method according to any one of claims 1 to 23, wherein the second period is at least one day.

25. The method according to any one of claims 1 to 24, wherein the second period is 4 to 7 days.

26. The method according to any one of claims 1 to 25, wherein the second period is 4 to 6 days.

27. The method according to any one of claims 1 to 26, wherein the second period is approximately 5 days.

28. The method according to any one of claims 1 to 27, wherein the first period is approximately 10 days and the second period is approximately 5 days.

29. A method for producing dulaglutide, a) A step of culturing mammalian cells expressing the dulaglutide in cell culture medium at a pH setting of approximately 6.86 with a dead zone of approximately 0.09 for a first period including 9 to 10 days, and thereafter, b) A step of culturing the mammalian cells in the cell medium at a pH setting of approximately 7.0 with a dead zone of approximately 0.05 for a second period including 4 to 5 days, A method wherein the dulaglutide is produced by the mammalian cells as a result.

30. The method according to any one of the prior claims, further comprising culturing the mammalian cells at a first temperature for the first 1 to 5 days of the first period, and then culturing the mammalian cells at a second temperature for 9 to 14 days.

31. The method according to claim 30, wherein the second temperature is lower than the first temperature.

32. The method according to claim 30 or 31, wherein the first temperature is 34°C to 40°C.

33. The method according to any one of claims 30 to 32, wherein the first temperature is approximately 36°C.

34. The method according to any one of claims 30 to 33, wherein the second temperature is 30°C to 36°C.

35. The method according to any one of claims 30 to 34, wherein the second temperature is approximately 33°C.

36. The method according to any one of claims 30 to 35, wherein the mammalian cells are cultured at the first temperature for 3 to 4 days.

37. The method according to any one of claims 30 to 36, wherein the mammalian cells are cultured at the first temperature for about three days.

38. The method according to any one of claims 30 to 37, wherein the mammalian cells are cultured at the second temperature for 11 to 12 days.

39. The method according to any one of claims 30 to 38, wherein the mammalian cells are cultured at the second temperature for about 11 days.

40. The method according to any one of the prior claims, wherein the cells are cultured in a bioreactor.

41. The method according to claim 40, wherein the mammalian cells are cultured in a fed-batch mode.

42. The method according to any one of the prior claims, further comprising adding a base source to the cell medium when the pH falls below the dead zone during the first period, or adding an acid source to the cell medium when the pH rises above the dead zone.

43. The method according to any one of the prior claims, further comprising adding a base source to the cell medium if the pH falls below the dead zone during the second period, or adding an acid source to the cell medium if the pH rises above the dead zone.

44. The aforementioned acid source is CO ２ The method according to claim 42 or 43.

45. The method according to any one of claims 42 to 44, wherein the base source is a solution containing NaOH.

46. The method according to any one of the prior claims, wherein the cell culture medium is spagged with air during the first period and / or the second period.

47. The method according to any one of the prior claims, wherein the mammalian cells are selected from the group consisting of COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 cells, PER. C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, B-W cells, L-M cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

48. The method according to any one of the prior claims, wherein the mammalian cells are CHO cells.

49. The method according to any one of the prior claims, wherein one or more proteases have lower activity in the second pH dead zone compared to the first pH dead zone.

50. The method according to any one of the prior claims, wherein one or more proteases have lower activity at the second pH setting compared to the first pH setting.

51. The method according to claim 50, wherein one or more proteases partially cleave dulaglutide at its N-terminus, and optionally the protease is cathepsin D.

52. The method according to any one of the prior claims, wherein less than about 4% of the dulaglutide produced is partially cleaved at the N-terminus.

53. The method according to claim 52, wherein the partially cleaved form of dulaglutide does not have an HG residue at the N-terminus of dulaglutide.

54. A method for producing dulaglutide in CHO cells, a) A step of culturing CHO cells for about 10 days in a cell medium maintained at a first pH setting of about 6.68 with a dead zone of about 0.09, and then b) A step of culturing CHO cells for about 5 days in a cell medium maintained at a second pH setting of about 7.0 having a dead zone of about 0.05, The aforementioned culturing process is started at a temperature of approximately 36°C, and then reduced to approximately 33°C after about 3 days. A method wherein the dulaglutide is produced by the CHO cells as a result.

55. The method according to claim 54, wherein the CHO cells are cultured in a bioreactor in a fed-batch mode.

56. NaOH is added to the cell culture medium when the pH falls below the dead zone of either the first pH setting or the second pH setting, and CO is added when the pH rises above the dead zone of either the first pH setting or the second pH setting. ２ The method according to claim 54 or 55, wherein the above is added to the cell culture medium.

57. The method according to any one of claims 54 to 56, wherein the cell culture medium is spared with air once or more times.

58. The method according to any one of claims 54 to 57, wherein less than 4% of the dulaglutide produced by the CHO cells is partially cleaved at the N-terminus.

59. dulaglutide produced by the method described in any one of the prior claims.

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